Bacteria of the genus Clostridium are gram positive and include many pathogenic species responsible for significant mortality and morbidity in both humans and animals. The genus Clostridium produces more protein toxins than any other bacterial genus. Clostridium tetani is a common soil dwelling organism which produces a neurotoxin responsible for the disease tetanus. Clostridium perfringens is a common etiological agent of gas gangrene and food poisoning. Clostridium difficile is a common cause of gastroenteritis and pseudomembraneous colitis, particularly among elderly hospital patients who have had their intestinal flora depopulated by treatment with antibiotics. Clostridium botulinum is a heterologous collection of clostridial strains that have the common property of producing a distinctive neurotoxin (BoNT) of extraordinary potency. This neurotoxin, also known as botulinum toxin, is the cause of the severe neuroparalytic illness in humans and animals referred to as botulism. Even in advanced societies, there are fatalities each year due to oral ingestion of the botulinum toxin produced by bacteria in contaminated food sources.
Because of their extreme toxicity the neurotoxins produced by Clostridium botulinum have been the subject of extensive study. Botulinum toxins are classified into seven serotypes, referred to as serotypes A through G, on the basis of their immunological properties. Botulinum Type A neurotoxin is among the most poisonous natural substances known to science. The amino acid sequences of the toxins have been deduced and compared. See, for example, Minton, "Molecular Genetics of Clostridial Neurotoxins," in Clostridial Neurotoxins, C. Montecucco (Ed.) Springer-Verlag, Berlin (1995).
Botulinum toxin type A has become an extremely important pharmaceutical for the treatment of segmental movement disorders, spasticity, pain syndromes, and various other neuronal disorders. Botulinum toxin specifically and tightly binds to cholinergic neurons. Upon endocytosis and internalization into the nerve terminal, the light chain of the toxin acts to block or slow the exocytotic release of neurotransmitters, particularly acetylcholine. Selective injection of botulinum toxin into neuromuscular regions produces a local weakening of proximal muscles and relief from excessive involuntary muscle contractions. In addition to directly affecting cholinergic neurotransmission, botulinum toxin also exerts other poorly understood effects including altering activity of autonomic ganglia. The outstanding properties of botulinum toxin as a pharmacological agent are its specificity for peripheral nerves and its long duration of action. Complications and drawbacks of botulinum toxin therapy include immunological resistance in some patients and diffusion and resulting ptosis of neighboring muscles. These side effects can be avoided by proper expression, purification and preparation of the toxin or toxin chains or fragments for pharmaceutical use. See Schantz and Johnson, Microbiological Reviews, 56:80-00 (1992).
During the past decade, interest in botulinum toxins has accelerated due to the discovery that the light chains of botulinum neurotoxins specifically cleave proteins in nerve terminals that are necessary for exocytosis and neurotransmission. Interest in botulinum toxins has also been triggered by the potential for the use of these toxins in bioterrorism and, as a result, government agencies are actively investigating countermeasures against them. A pentavalent vaccine has been developed which is effective against serotypes A through E, but this vaccine is not effective against types F and G. However, the manufacturing site for this vaccine has been closed since it did not meet U.S. FDA requirements. Also, the pentavalent vaccine has drawbacks in that it can cause acute adverse reactions in some of the recipients.
Recently the U.S. Department of Defense has made an effort to produce recombinant vaccines in which fragments of botulinum toxin are produced in heterologous hosts. However, it has been found that the toxins are produced very poorly in E. coli, yeast, and other potential hosts. Scientists have entirely resynthesized the genes for certain serotypes to achieve better expression, but they have found that the recombinant proteins can undergo posttranslational modifications in heterologous hosts.
Botulinum toxins are found natively both in bacterial cultures and in contaminated foods complexed with other nontoxic proteins including nontoxic nonhemagglutinin (NTNH) and several hemagglutinins (HA). The neurotoxin component of the toxin complex is a 150 kDa protein comprising a heavy (HC) and a light (LC) chain. The LC contains the catalytic domain that cleaves nerve proteins essential for neurotransmission. Separation of purified toxins, or toxin domains, or toxin fragments is possible, but is difficult, laborious, and the yields are low. The clinical use of purified botulinum toxin fragments is complicated by the need for extreme care of purification, since small amounts of any contaminating active toxin can be highly dangerous. Biochemical preparations of toxin chains or fragments are always contaminated with low levels of active neurotoxin.
Two main strategies have been utilized to obtain clostridial neurotoxins, individual chains of the toxins, or non-toxigenic components of the toxin complex. The first strategy is to isolate the desired protein itself from cultures of the toxigenic C. botulinum strain. The second strategy is to produce the toxin or toxin fragments in heterologous hosts.
Unfortunately, the heterologous expression of clostridial genes in heterologous hosts has been found to be quite inefficient. Available information on clostridial gene expression in E. coli in particular, and also other heterologous hosts, indicates that the expression of clostridial genes in these hosts occurs at very low levels and is relatively inefficient. This inefficiency arises, in part, from a striking characteristic of clostridial DNA in that it has an extremely low percentage G+C content, ranging typically from 20 to 29% for toxigenic species. Oddly, the percentage G+C content of the coding regions of the clostridial genome is consistently higher than that of non-coding intergenic regions. The extremely low percentage G+C content of clostridial DNA affects the pattern of codon usage which can effect protein production in heterologous hosts which are biased toward codons in which A and T predominate. This same pattern of codon usage has been reported in many clostridial species. Because of the striking differences in codon usage, available evidence suggests that clostridial genes are inefficiently expressed in other hosts, such as E. coli, primarily at the translational rather than transcriptional level, although factors other than codon bias also affect expression of protein toxins. Attempts have been made to overcome this low expression problem by re-synthesizing the gene to incorporate codons preferred by E. coli or other hosts such as yeast, or to use an expression host that prefers low percentages G+C content, such as Lactococcus species. These strategies have been somewhat successfully used with the tetanus toxin.
Expression of clostridial genes in native clostridial species is, as might be expected, more efficient and the resulting proteins would be less prone to have structural or sequence errors and would undergo proper posttranslational modifications. However, prior methods for the transfer of genes into or amongst clostridial bacteria are either inefficient or non-existent. No shuttle vectors are currently available which have been shown to provide efficient gene transfer to C. botulinum. Also, handling of and culturing of these bacteria is difficult since not only are they highly toxic, the organisms are obligate anaerobes which die if exposed to oxygen. Therefore, the clostridia must be handled under specialized conditions. These technical difficulties limit the approaches that can be used for gene transfer in other bacteria such as electroporation, transformation and transduction.
Accordingly, the study and the production of clostridial toxin genes, as well as other classes of clostridial genes, would be greatly facilitated by a system that permits the introduction and expression of clostridial genes in a host of that genus which does not otherwise produce, at least prior to transformation, a toxin or toxin protein.